Engineered DNA Polymerases

Abstract

This chapter reviews the methods used to generate variants of DNA polymerases that have improved ability, in particular to accept unnatural nucleotides, focusing especially on in vitro and directed evolution methods. Several natural families of DNA polymerases have independently evolved for millions of years to accept their natural nucleotide substrates with high fidelity and the ability to exclude closely related structures, such as ribonucleoside derivatives. However, polymerases that can accept unnatural nucleotide substrates would have many applications in biotechnology. Directed evolution may be an efficient method to produce new DNA polymerases capable to do so. Directed evolution relies on methods to create a library of sequence diverse polymerases starting with a gene for a parent polymerase. These methods are reviewed here, as well as examples of their application to produce variant polymerases. An evolutionary rationalization is offered to explain some mutations produced by directed evolution experiments.